An approach of introducing a foreign gene using an Agrobacterium-derived plasmid has been known widely as one of processes for generating a transformed plant by introducing a foreign gene, in such a case when a target plant undergoes infection with the pathogenic bacterium Agrobacterium (cancer bacterium). A Ti-plasmid (Tumor inducing plasmid) from Agrobacterium tumefaciens or a Ri-plasmid from Agrobacterium rhizogenes has been studied widely as the Agrobacterium-derived plasmid that can be used in genetic recombination. These Agrobacterium-derived plasmids, Ti- and Ri-plasmids, contain a region called T-DNA. This T-DNA region is incorporated into plant chromosomes. Moreover, gene clusters involved in the incorporation of the T-DNA region into plant chromosomes are present in a Vir region in proximity to the T-DNA region. The Vir region contains VirA, VirB, VirC, VirD, VirE, and VirG genes, and products thereof have been understood to promote the introduction of the T-DNA region into plant chromosomes.
The T-DNA region is approximately 20 kb in size. Although the structure of the region differs among species, 25-base-pair sequences at both ends of this T-DNA region are similar among species and essential for the incorporation of T-DNA into chromosomal DNA. A gene of interest is inserted into a site flanked by the 25-base-pair sequences (border nucleotide sequences) at both ends of the T-DNA region. The modified Ti-plasmid is introduced into Agrobacterium to generate a transformant. A target plant is infected with this transformant such that the T-DNA region comprising the insert of the gene of interest is introduced into the chromosomal DNA of the target plant with high efficiency.
A gene encoding a Bacillus thuringiensis-derived insecticidal protein (BT-toxin) has been introduced into the chromosomal DNA of a Nicotiana tabacum cell by using the Ti-plasmid system via infection with an Agrobacterium transformant to generate a transformed plant that recombinantly expresses the BT-toxin therein. Furthermore, a gene encoding firefly-derived luminescent protein (luciferase) has been introduced into the chromosomal DNA of a Nicotiana tabacum cell by using the Ti-plasmid system to generate a transformed plant that expresses an active luciferase therein.
It has been reported that the introduction of T-DNA into the chromosomal DNA of a target plant using a Ti-plasmid system via infection with an Agrobacterium transformant usually takes place with frequency on the order of 1 to 2 copies per plant individual (see Non-Patent Document 1: Azpiroz-Leehan, R. et al., Trends Genet., 13 (4), p. 152-6 (1997)). Moreover, the introduction of T-DNA into chromosomal DNA is due to such a mechanism of homologous replacement-based DNA insertion using the 25-base-pair sequences at both ends of the T-DNA region. Accordingly, a T-DNA fragment can be inserted at random into a large number of sites having a nucleotide sequence that permits the homologous replacement-based DNA insertion, which are present in the chromosomal DNA of the target plant. The fact that the introduction of T-DNA usually takes place with frequency on the order of 1 to 2 copies per plant individual means that the insertion of T-DNA takes place only at one site, or two sites at most, of the large number of sites that permit insertion, per plant individual.
Such adavantage that the introduction of T-DNA usually takes place with frequency on the order of 1 to 2 copies per plant individual has been employed profitably to develop a Fox-hunting system (full-length cDNA over-expression gene hunting system) (see Patent Document 1: pamphlet of WO 03/018808 and Non-Patent Document 2: Seki M., et al., Science Vol. 296, No. 5565, p. 141-145 (2002)). In a recombinant expression vector system used in the Fox-hunting system,
a T-DNA region thereof, which is finally introduced into the chromosomal DNA of the plant, comprises:
as regulatory sequences for the expression of an inserted gene, a “cassette” comprising a combination of a promoter sequence that induces expression in a target plant and a terminator sequence that terminates transcription; and
a cloning site for inserting full-length cDNA derived from the gene to be inserted, which is provided downward of the promoter sequence and upward of the terminator sequence. An E. coli/Agrobacterium binary vector, for example, pTAS- or pBig-derived vector, is used, which comprises said T-DNA region and a Ti-plasmid-derived replication origin (ORI) and is additionally equipped with a selection marker gene such as an antibiotic resistance gene and further with an E. coli plasmid-derived replication origin (ORI). The full-length cDNA derived from the gene to be introduced is inserted using the cloning site in the binary vector to construct a vector for transformation, which vector is in turn introduced into Agrobacterium having a Ti-plasmid to prepare an Agrobacterium transformant. A target plant is infected with this Agrobacterium transformant using, for example, a floral dipping method, such that the T-DNA region in the binary vector retained in the transformant is introduced into the chromosomal DNA of the target plant. Each plant individual that has undergone the dipping treatment is subjected to self-pollination, and then a T1 seed is harvested from the plant individual. The T-DNA is inserted only with frequency on the order of 1 to 2 copies in the chromosomal DNA of the target plant. Therefore, when pollen or ovule cells having each n chromosome are formed from 2n homologous chromosomes (a*/a type) by meiosis, the ratio between pollen or ovule cells (a* type) in which the T-DNA has been introduced and pollen or ovule cells (a type) in which the T-DNA has not been introduced is almost equal in number. Accordingly, among T1 seeds, those retaining the T-DNA introduced in the chromosomal DNA (a*/a type or a*/a* type) and those not retaining the T-DNA (a/a type) coexist according to the Mendel's laws of heredity. Whether or not the T-DNA region is actually retained in the chromosomal DNA is confirmed by germinating the harvested T1 seed on, for example, a selective medium supplemented with a drug, and performing primary screening based on the presence or absence of expression of the marker gene such as a drug resistance gene equipped in advance to the T-DNA region to select a transformed plant, in which the T-DNA region has been introduced.
Furthermore, the germinated seed selected by primary screening is grown, and the plant grown therefrom is subjected to phenotypic screening for selecting a transformed plant that exhibits difference in phenotype, which is associated with the expression of the gene inserted in the T-DNA region, in comparison with that of a wild-type plant. For the transformed plant selected in this phenotypic screening, a PCR product derived from the T-DNA region is prepared using a PCR method to verify that the T-DNA region comprising the insert of the full-length cDNA derived from the gene to be introduced is indeed present in the chromosomal DNA thereof. Moreover, each individual of the verified transformed plant selected in phenotypic screening is subjected to self-pollination, and then a T2 seed is harvested from the plant individual.
The approach for preparing a transformed plant using the T-DNA recombinant expression vector system, which has been employed in the Fox-hunting system, can be applied to various plants which are capable of undergoing Agrobacterium infection and producing a harvestable T1 seed through the self-pollination of each plant individual.
The plants to which the approach can be applied includes plants belonging to the family Brassicaceae, Poaceae, Soianaceae, or Leguminosae, for instance, a typical example thereof includes the following plants:    the family Brassicaceae: Arabidopsis thaliana     the family Solanaceae: Nicotiana tabacum     the family Poaceae: Zea mays, Oryza sativa     the family Leguminosae: Glycine max. 
Patent Document 1: WO 03/018808 A1
Non-Patent Document 1: Azpiroz-Leehan, R. et al., Trends Genet., 13 (4), p. 152-6 (1997)
Non-Patent Document 2: Seki M., et al., Science Vol. 296, No. 5565, p. 141-145 (2002)